The primary transcriptome, small RNAs, and regulation of ... · 85 Cells were grown overnight in 5 mL Lennox (L-) broth (10 g L-1. tryptone, 5 g L-1. yeast 86 extract, 5 g L-1. NaCl;

The primary transcriptome, small RNAs, and regulation of 1 antimicrobial resistance in Acinetobacter baumannii 2

Carsten Kröger1*, Keith D. MacKenzie2, Ebtihal Y. Al-Shabib2, Morgan W. B. Kirzinger2, Danae 3 M. Suchan2, Tzu-Chiao Chao2, Valentyna Akulova2, Alesksandra A. Miranda-CasoLuengo1, 4 Vivian A. Monzon1, Tyrrell Conway5, Sathesh K. Sivasankaran6, Jay C. D. Hinton3, Karsten 5 Hokamp4, and Andrew D. S. Cameron2* 6

1 Department of Microbiology, School of Genetics & Microbiology, Moyne Institute of 7 Preventive Medicine, Trinity College Dublin, Dublin 2, Ireland 8

2 Institute for Microbial Systems and Society, Department of Biology, University of Regina, 9 Regina, Saskatchewan, S4S 0A2, Canada. 10

3 Institute of Integrative Biology, University of Liverpool, Liverpool L69 7ZB, UK. 11 4 Department of Genetics, School of Genetics & Microbiology, Smurfit Institute of Genetics, 12

Trinity College Dublin, Dublin 2, Ireland 13 5 Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, 14

OK 74078, USA 15 6 John P. Hussman Institute for Human Genomics, Miller School of Medicine, University of 16

Miami, Miami, Florida, USA 17

Correspondence: [email protected] & [email protected] 18

Key words 19 Acinetobacter baumannii 20 Differential (d)RNA-seq 21 Small RNA 22 Antibiotic resistance 23 craA efflux pump 24

ABSTRACT 25

We present the first high-resolution determination of transcriptome architecture in the 26 priority pathogen Acinetobacter baumannii. Pooled RNA from 16 laboratory conditions was used 27 for differential RNA-seq (dRNA-seq) to identify 3731 transcriptional start sites (TSS) and 110 28 small RNAs, including the first identification in A. baumannii of sRNAs encoded at the 3′ end of 29 coding genes. Most sRNAs were conserved among sequenced A. baumannii genomes, but were 30 only weakly conserved or absent in other Acinetobacter species. Single nucleotide mapping of 31 TSS enabled prediction of –10 and –35 RNA polymerase binding sites and revealed an 32 unprecedented base preference at position +2 that hints at an unrecognized transcriptional 33 regulatory mechanism. To apply functional genomics to the problem of antimicrobial resistance, 34 we dissected the transcriptional regulation of the drug efflux pump responsible for 35 chloramphenicol resistance, craA. The two craA promoters were both down-regulated >1000-fold 36

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when cells were shifted to nutrient limited medium. This conditional down-regulation of craA 37 expression renders cells sensitive to chloramphenicol, a highly effective antibiotic for the treatment 38 of multidrug resistant infections. An online interface that facilitates open data access and 39 visualization is provided as “AcinetoCom” (http://bioinf.gen.tcd.ie/acinetocom/). 40

INTRODUCTION 41

In 2017, the World Health Organization (WHO) ranked carbapenem-resistant A. baumannii a top 42 priority critical pathogen in the WHO’s first-ever list of priority antimicrobial-resistant pathogens 43 (1). It is estimated that one million A. baumannii infections occur worldwide each year, causing 44 15,000 deaths (2). In the United States, A. baumannii is responsible for more than 10% of 45 nosocomial infections, and causes a variety of diseases such as ventilator-associated pneumonia, 46 bacteraemia, skin and soft tissue infections, endocarditis, urinary tract infections and meningitis 47 (3). Despite the urgency to develop new antimicrobial drugs, we know little about A. baumannii 48 infection biology and virulence mechanisms because only a few A. baumannii virulence genes and 49 their regulation have been functionally studied (4). 50

Characterizing the transcriptional landscape and simultaneously quantifying gene 51 expression on a genome-wide scale using RNA sequencing (RNA-seq) is a cornerstone of 52 functional genomic efforts to identify the genetic basis of cellular processes (5). Given that RNA-53 seq identifies transcripts for the entire genome at single-nucleotide resolution in a strand-specific 54 fashion, it is the ideal tool to uncover transcriptome features. Recent technical enhancements to 55 RNA-seq enable improved delineation of transcriptional units to localize transcriptional start sites, 56 transcript ends, sRNAs, antisense transcription and other transcriptome features (5, 6). The ability 57 to quantify gene expression and characterize transcripts from any organism is particularly valuable 58 for the study of emerging bacterial pathogens, where research knowledge lags behind the global 59 spread, health burden, and economic impacts of disease. 60

To address the lack of basic biological insight of A. baumannii, we used a functional 61 genomics approach to investigate the transcriptome architecture of A. baumannii ATCC 17978 62 and to study virulence gene expression and antibiotic resistance in this priority pathogen. A. 63 baumannii ATCC 17978 is one of the best-studied strains of Acinetobacter, and is a useful model 64 for genetic manipulation due to its natural sensitivity to most antibiotics used in the laboratory (7, 65 8). Differential RNA-seq (dRNA-seq) facilitates the precise identification of TSS by 66 distinguishing 5’-triphosphorylated (i.e. newly synthesized, “primary” transcripts) and other 5’-67 termini (such as processed or dephosphorylated) of RNA species (9). dRNA-seq analysis of 68 transcription in virulence-relevant conditions, antimicrobial challenge, and standard laboratory 69 culture generated a high-resolution map of TSS and small RNAs in the A. baumannii chromosome, 70 revealing the precise location of 3731 promoters and 110 small RNAs. This comprehensive view 71 of TSS identified two promoters that regulate chloramphenicol resistance by the CraA efflux pump 72 and we discovered growth conditions that stimulate low craA expression and render A. baumannii 73 sensitive to chloramphenicol. To facilitate similar discoveries by the research community, we 74



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created the online resource AcinetoCom (http://bioinf.gen.tcd.ie/acinetocom/) for free and 75 intuitive exploration of the A. baumannii transcriptome. 76

MATERIALS AND METHODS 77

Bacterial strains and growth conditions. 78

Acinetobacter baumannii ATCC 17978 was obtained from LGC-Standards-ATCC. We used the 79 reference strain containing all three plasmids (pAB1, pAB2, pAB3) for dRNA-seq analysis. RNA-80 seq revealed that pAB3 was absent in the strain used for early stationary phase transcriptome 81 analysis, indicating that pAB3 was lost during shipping of ATCC 17978 between our laboratories. 82 Comparing RNA-seq reads to the reference genome did not detect any point mutations in the 83 derivative strain lacking pAB3. 84

Cells were grown overnight in 5 mL Lennox (L-) broth (10 g L-1 tryptone, 5 g L-1 yeast 85 extract, 5 g L-1 NaCl; pH 7.0), sub-cultured the next morning (1:1,000) in 250 ml flasks containing 86 25 ml L-broth, and grown at 37°C with shaking at 220 RPM in a waterbath. Early stationary phase 87 (ESP) samples were collected when cultures reached an OD600 of 2.0 in L-broth. Cell cultures were 88 exposed to several additional growth conditions and environmental shocks to induce gene 89 expression and obtain RNA for the “pool sample”; these conditions are described in the subsequent 90 sentences and summarized in Table 1. In addition to sampling colonies from L-agar plates 91 incubated at 37˚C, cells were sampled at select culture densities during growth in L-broth in 92 shaking flasks (OD600 values of 0.25, 0.75, 1.8, 2.5 and 2.8). Exponentially-growing cultures 93 (OD600 of 0.3) were exposed to different environmental shocks for 10 minutes, including 250 µL 94 of a 1:100 dilution of disinfectant (Distel 'High Level Medical Surface Disinfectant', non-95 fragranced), hydrogen peroxide to a final concentration of 1 mM, ethanol to a final concentration 96 (v/v) of 2%, kanamycin to a final concentration of 50 µg mL-1, NaCl to a final concentration of 97 0.3 M or 2,2’-dipyridyl to a final concentration of 0.2 mM. Cells were also cultured in M9 minimal 98 medium containing 1 mM MgSO4, 0.1 mM CaCl2 and a carbon source of either 31.2 mM fumarate 99 or 33.3 mM xylose. To mimic growth in environmental conditions, cells were grown at low 100 temperatures (25˚C) until an OD600 of 0.3. Additional cells were also grown at 25˚C to an OD600 101 of 0.3 before cultures were switched to growth at an ambient temperature of 37˚C for 10 minutes. 102

RNA extraction, ribosomal RNA depletion, DNase digestion and the RNA pool. 103

Total RNA was isolated from A. baumannii ATCC 17978 cells using TRIzol as described 104 previously for Salmonella enterica serovar Typhimurium (10). The RNA quality was analyzed 105 using an Agilent Bioanalyzer 2100 and RNA concentration was measured on a NanoDrop™ 106 spectrophotometer or the Qubit™ (Invitrogen). The ESP RNA samples were depleted for 107 ribosomal RNA using the RiboZero rRNA Removal Kit Bacteria (Illumina), and contaminating 108 DNA was digested with DNaseI using the TURBO DNA-free™ kit (Ambion). To generate the 109 RNA pool, equal amounts of RNA from each condition were combined into a single sample. 110



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Library preparation for next generation sequencing. 111

Libraries from three biological replicates of the ESP condition were prepared using the 112 NEBNext™ Ultra RNA Library Prep Kit for Illumina (NEB) according to the manufacturer’s 113 instructions. These libraries were sequenced on an Illumina MiSeq (paired-end reads). The 114 libraries of the pooled RNA sample for dRNA-seq were prepared by vertis Biotechnologie AG 115 (Freising, Germany) and sequenced on an Illumina NextSeq machine (single reads). For dRNA-116 seq, RNA samples were digested with Terminator Exonuclease (TEX) prior to cDNA library 117 preparation (9, 11). We noted that for highly-expressed genes in the ESP samples, there were 118 distinct transcripts mapped to the opposite strand that perfectly mirrored the expected transcript. 119 This is best explained by incomplete depletion of non-template strand (second strand incorporated 120 dUTP) by the USER enzyme during library preparation with NEBNext™ Ultra. Therefore, we 121 considered only the pooled RNA-seq data from Vertis Biotechnologie AG, which is generated by 122 an RNA adaptor ligation step before cDNA synthesis, to determine the amount of antisense 123 transcription. 124

Mapping of sequencing reads, statistics and analysis. 125

The original genome sequence for A. baumannii ATCC 17978 identified two plasmids (pAB1 and 126 pAB2) (7). However, it was subsequently revealed that ATCC 17978 carries a third plasmid 127 (pAB3) that was incorrectly assembled as chromosome sequence in the first assembly (12), thus 128 the A. baumannii ATCC 17978-mff genome (accession: NZ_CP012004.1) was used as a reference 129 sequence in our study. DNA sequencing reads were mapped using bowtie2 with the –local –very-130 sensitive settings for use of soft-clipping and maximum sensitivity (13). Conversion into BAM 131 and BigWig files was carried out using SAMtools (14) and Galaxy at galaxy.org (15). Mapped 132 reads were visualized in the Integrated Genome Browser (16) and Jbrowse (17). Number of 133 sequence reads mapped were (the underscore indicates the biological replicate) ESP_1 134 (10,588,403; mapped uniquely: 8,403,658), ESP_2 (9,374,114; mapped uniquely: 6,769,516), 135 ESP_3 (11,121,847; mapped uniquely: 7,891,371, RNA_pool (66,853,984: mapped uniquely: 136 19,607,931) and dRNA-seq_Pool (77,237,102; mapped uniquely: 30,041,320). The difference in 137 percentage of uniquely mapped reads of the ESP conditions and the Pool samples is caused by the 138 fact that RNA from the ESP condition was depleted for rRNA, while the RNA samples of the RNA 139 Pool were not. FeatureCounts was used to summarize read counts for each annotated feature (18). 140 These values were transformed into transcripts per million reads (TPM) following the formula 141 provided by Li et al. (19); (Table S4). 142

Data availability 143

The raw data and normalized mapped reads are available from the Gene Expression Omnibus 144 (GEO) at accession no: GSE103244. 145



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Identification and categorization of transcriptional start sites (TSS). 146

Before the identification of TSS, the coverage files for the RNA Pool and TEX-digested RNA Pool 147 sample were normalized to account for different library sizes. The number of mapped nucleotides 148 for each position was divided by the number of uniquely mapped reads of the smallest library 149 (ESP_2) and then multiplied by the number of reads for the library being normalized. The locations 150 of TSS were predicted with TSSpredator (20) using the default settings with the exception of 151 changing the step height option to 0.2, the 5′ UTR length to 400 and the enrichment factor to 1.5. 152 The locations of the predicted TSS were manually inspected in IGB and Jbrowse. TSS that exist 153 ≤400 nt upstream of the start of a coding region and TSS of non-coding RNA genes were classified 154 as Primary or Secondary TSS; in cases where multiple candidate TSS are upstream, the TSS 155 associated with the strongest expression was designated as the Primary TSS, while all additional 156 TSS were designated Secondary TSS. TSS with intragenic location on the sense strand were 157 classified as Internal TSS and when located on the antisense strand as Antisense TSS. TSS were 158 classified as Orphan if they could not be assigned to any other category. Occasionally, manual 159 curation changed the classification of TSS, e.g. five genes with a 5’UTR >400 nt were classified 160 as Primary or Secondary (Table S2). 161

Identification of –10 and –35 sigma factor motifs. 162

DNA sequences upstream of all transcription start sites were extracted in two blocks of 163 nucleotides: between position –3 to –18 (–10 block) and between –16 to –50 (–35 block). Each 164 block was searched with the unbiased motif finding program MEME 4.11.2 (21). Each sequence 165 block was searched with the following settings: one occurrence per sequence with a width of 6-12 166 bp (–10 motif) or 4-10 bp (–35 motif). Sequence logos were generated by WebLogo (22). 167

Identification of candidate sRNA genes and prediction of transcription terminators. 168

The identification of candidate sRNA genes was carried out manually in IGB and Jbrowse. A new 169 candidate sRNA gene was assigned if a short (<500 nt) unannotated transcript was observed in the 170 RNA-seq data. The borders of the transcript were informed by the location of TSS determined by 171 dRNA-seq data and the location of predicted Rho-independent transcription terminators using 172 ARNold with the default setting (23). 173

sRNA conservation analysis. 174

The conservation of candidate sRNA genes was investigated using GLSEARCH (24). The genome 175 sequences were obtained from NCBI (Acinetobacter baumannii Ab04-mff (NZ_CP012006.1), 176 Acinetobacter baumannii AB030 (NZ_CP009257.1), Acinetobacter baumannii AB0057 177 (NC_011586.1), Acinetobacter baumannii AB5075-UW (NZ_CP008706.1), Acinetobacter 178 baumannii ACICU (NC_010611.1), Acinetobacter baumannii strain ATCC 17978 179 (NZ_CP018664.1), Acinetobacter baumannii ATCC 17978-mff (NZ_CP012004.1), 180 Acinetobacter baumannii str. AYE (NC_010410.1), Acinetobacter baumannii IOMTU 433 181



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(NZ_AP014649.1), Acinetobacter baumannii LAC-4 (NZ_CP007712.1), Acinetobacter 182 baumannii A1 (NZ_CP010781.1), Acinetobacter baumannii USA15 (NZ_CP020595.1), 183 Acinetobacter baumannii XH386 (NZ_CP010779.1), Acinetobacter baumannii TYTH-1 184 (NC_018706.1), Acinetobacter baumannii XDR-BJ83 (NZ_CP018421.1), Acinetobacter baylyi 185 DSM 14961 (NZ_KB849622.1-NZ_KB849630.1), Acinetobacter equi 114 (NZ_CP012808.1), 186 Acinetobacter haemolyticus CIP 64.3 (NZ_KB849798.1-KB849812.1), Acinetobacter lwoffii 187 NCTC 5866 (NZ_KB851224.1-NZ_KB851239.1), Acinetobacter nosocomialis 6411 188 (NZ_CP010368.1), Acinetobacter pittii PHEA-2 (NC_016603.1), Acinetobacter venetianus VE-189 C3 (NZ_CM001772.1), Moraxella catarrhalis BBH18 (NC_014147.1), Pseudomonas aeruginosa 190 PAO1 (NC_002516.2), Pseudomonas fluorescens F113 (NC_016830.1), Pseudomonas putida 191 KT2440 (NC_002947.4), Pseudomonas syringae pv. syringae B728a (NC_007005.1). The score 192 shows percentage sequence identity with the reference sequence (A. baumannii 17978-mff). 193

Northern blotting. 194

Northern blotting was performed using the DIG Northern Starter Kit (Roche) using DIG-labelled 195 riboprobes generated by in vitro transcription as described earlier and according to the 196 manufacturer’s instructions (10). Templates for in vitro transcriptions using T7 RNA polymerase 197 were generated by PCR using DNA oligonucleotides listed in Table S1. Five μg of total RNA was 198 separated on a 7% urea-polyacrylamide gel in 1x Tris-borate-EDTA (TBE) buffer cooled to 4°C. 199 Band sizes on the Northern blots were compared with Low Range single strand RNA ladder 200 (NEB). After blotting and UV crosslinking, the lane with the ladder was cut off, stained with 0.1% 201 methylene blue solution for 1 min at room temperature, destained in sterile water, and re-attached 202 to the membrane before detection of chemiluminescent bands using an ImageQuant LAS4000 203 imager (GE Healthcare). 204

Chloramphenicol sensitivity and craA expression analysis. 205

Cells were cultured overnight in L-broth containing 25 µg mL-1 chloramphenicol and the next 206 morning inoculated to OD600 0.05 in 20 mL fresh medium and grown to OD600 of 0.3 in a 250-mL 207 flask with shaking at 37 °C. Cells were captured on 0.2 µm analytical test filter funnels (Nalgene), 208 washed with M9 containing 20 mM pyruvate as the carbon source and the filter (retaining the cells) 209 was transferred to M9 plus pyruvate. The cells were washed off the filter and inoculated in fresh 210 20 mL M9 plus pyruvate medium at an OD600 of 0.1 and incubated at 37°C with shaking for 5 211 days. Sampling for qPCR and chloramphenicol resistance was performed at t = 0, 4h, 24h, 48h, 212 and 96h and transcription was stopped by the addition of phenol:ethanol stop solution (25, 26). 213 For qPCR, total RNA was isolated from cultures using the EZ-10 Spin Column Total RNA Mini-214 Preps Super Kit (BioBasic) and purity and quality was assessed by 1% agarose gel electrophoresis. 215 For each sample, 5 µg total RNA was DNase treated in a 50 μL reaction using the Turbo DNA-216 free kit (AMBION), and cDNA templates were synthesized by random priming 0.5 µg RNA in a 217 20 µL reaction using Verso cDNA synthesis kit (Fisher). PCR reactions were carried out in 218 duplicate with each primer set on an ABI StepOnePlus PCR System (Applied Biosystems) using 219



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Perfecta SYBR Fastmix with ROX (Quanta Biosciences). Standard curves were included in every 220 qPCR run; standard curves were generated for each primer set using five serial ten-fold dilutions 221 of A. baumannii genomic DNA. Quantitative PCR (qPCR) primers are listed in Table S1. Viable 222 counts were performed by serial dilution and drop-plating on L-agar plates without or with 223 chloramphenicol (10 µg mL-1); colony forming units were counted after overnight incubation at 224 37°C. 225

RESULTS AND DISCUSSION 226

Global identification of A. baumannii transcriptional start sites 227

To generate a comprehensive list of transcriptional start sites for A. baumannii, we adopted the 228 technique developed for Salmonella Typhimurium in which diverse laboratory conditions were 229 used to elicit transcription activation from as many gene promoters as possible (11). Thus, we 230 selected 16 conditions that reflect the environments experienced by A. baumannii during infection, 231 environmental survival, and general stress (Table 1). Specifically, A. baumannii was cultured in 232 L-broth and sampled throughout growth at OD600 0.25, 0.75, 1.8, 2.5, 2.8, in L-broth at 25°C, in 233 liquid M9 minimal medium with different sole carbon sources (xylose, fumarate), and grown on 234 an L-agar plate for 16 hours. We also isolated RNA from several “shock” conditions, including 235 disinfectant shock, osmotic shock (addition of sodium chloride), limitation of iron availability 236 (addition of 2,2’-dipyridyl), translational stress (addition of kanamycin), oxidative stress (addition 237 of hydrogen peroxide), temperature shift from ambient to body temperature (25 °C to 37 °C), and 238 the addition of ethanol (which increases virulence in A. baumannii infection of Dictyostelium 239 discoideum and Caenorhabditis elegans (27)). Total RNA was isolated from the 16 conditions, 240 pooled, and used as a single sample. 241

To enrich for primary transcripts, the pooled sample was digested with Terminator 242 Exonuclease (TEX) (9). Over 20 million mapped sequencing reads were analyzed with 243 TSSpredator software (20) and manually inspected with Integrated Genome Browser (IGB) and 244 Jbrowse (16, 17) to define the genomic location of 3731 TSS (Fig. 1). In addition, three biological 245 replicates of RNA isolated from OD600 2.0 (early stationary phase (ESP)) yielded over 6.7 million 246 mapped reads for each of the ESP biological replicates and the >20 million mapped reads for the 247 RNA pool provided sufficient depth to cover the bacterial transcriptome (28). 248

TSS were categorized according to their locations relative to coding sequences (Fig. 2A). 249 Of 3731 TSS, 1079 (~29%) were primary and 153 (~4%) were secondary TSS (Fig. 2B and Table 250 S2). Manual curation of the TSS predictions determined that five TSS had a 5′ UTR longer than 251 400 nt; these likely represent unusually long 5′ UTRs, but could encode small open reading frames 252 missed during automated gene annotation (Fig. 2C). About 18% (679) of transcripts were initiated 253 from inside of coding regions (i.e. internal TSS). An internal TSS can drive expression of 254 downstream genes, e.g. the promoter of DNA gyrase B-encoding gene (gyrB) lies within the ORF 255 located upstream encoding DNA recombination protein RecF. The biological role of internal 256 promoters that do not drive the expression of a downstream gene is unclear, however, a subset 257 initiates the expression of small RNAs located at the 3’-end of coding genes (see below). 258



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TSSpredator predicted that almost half (~47%, 1764/3731) of the identified TSS are located 259 antisense of coding regions. Manual inspection revealed that the large majority of antisense TSS 260 produce very short (<100 nt) and low-copy transcripts. The function of these short antisense 261 transcripts is debated and a portion might be considered as non-functional transcripts that are 262 initiated at promoter-like sequences (29, 30). However, an increasing number of studies suggest 263 that pervasive transcription (including short antisense transcripts) exhibit biological roles (31). The 264 ~1.5% (56/3731) of TSS that do not have a clear association with an annotated genomic feature 265 were categorized as orphan TSS. 266

A. baumannii promoter architecture 267

To initiate transcription, a sigma factor directs binding of RNA polymerase to DNA within 50 nt 268 upstream of a TSS (32). We determined the consensus promoter structure upstream of the 3731 269 TSS using unbiased motif searching to detect TSS-proximal and TSS-distal motifs corresponding 270 to the –10 and –35 locations of sigma factor binding sites. Because the spacing between –10 and 271 –35 binding sites differs between promoters, we searched for each motif separately; constraining 272 a specific spacing between –10 and –35 elements prevents resolution of the weaker motifs located 273 around the –35 position. MEME searching identified canonical RpoD (sigma 70) binding site 274 motifs in both the –10 and –35 locations (Fig. 2D), consistent with the expectation that RpoD is 275 responsible for a large majority of transcription initiation in A. baumannii. Aligning all 3731 A. 276 baumannii TSS revealed that adenine is the most common initiating nucleotide (A=47 %) (Fig. 277 2C). The majority of transcripts initiate with a purine nucleotide triphosphate (ATP= 47 %, GTP= 278 23 %); these are the primary energy storage molecules and thus provide a mechanism to regulate 279 transcription initiation. Yet it is position +2 that has the strongest sequence bias (T=57 280 %). Pyrimidine nucleotides are less abundant in cells, and the preference for pyrimidine 281 nucleotides at the –1 (72 % C+T) and +2 (81 % C+T) positions immediately flanking the TSS may 282 represent a mechanism that reduces accidental transcription initiation from these flanking positions 283 (10). 284

Untranslated regions (UTR) of mRNA perform multiple biological functions, including 285 providing binding sites for ribosomes and the potential to fold into mRNA secondary structures 286 that regulate transcription and RNA stability (33). The median length of 5′ UTR for primary TSS 287 is 37 nucleotides and secondary TSS is 100 nt (Fig. 2C). Similar UTR analyses have not been 288 performed for the E. coli transcriptome; the median length of 5′ UTR in Salmonella Typhimurium, 289 another Gamma-proteobacterium, is 55 nt for primary TSS and 124 nt for secondary TSS (10). 290

Identification of A. baumannii ATCC 17978 small RNAs 291

Small, regulatory RNAs are a class of post-transcriptional regulators that modulate gene 292 expression of many cellular processes (34, 35). Functions have been determined for some sRNAs 293 in Enterobacteriaceae (e.g. E. coli, Salmonella Typhimurium), but no sRNA functions have been 294 experimentally determined in A. baumannii (4). Two previous studies used bioinformatic and 295 experimental approaches to locate small RNAs in A. baumannii. In A. baumannii MTCC 1425 296



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(ATCC 15308), 31 sRNAs were predicted bioinformatically, of which three of which were verified 297 by Northern blotting (36). In A. baumannii AB5075, RNA-seq analysis of exponential growth in 298 lysogeny broth (LB) identified 78 sRNAs, six of which were verified by Northern blotting (37). 299 Here, we employed dRNA-seq on pooled growth conditions for de novo detection of sRNA 300 candidates; pooled RNA samples coupled with the high resolution determination of TSS has been 301 shown to identify a greater number of sRNA per genome compared to traditional methods (11). 302 Using the search criteria described in Material and Methods, sRNAs could occur within intergenic 303 regions, antisense to known coding regions, or within the 3′ end of mRNA. dRNA-seq provided 304 the necessary resolution to detect TSS within the 3′ end of coding genes. We identified a total of 305 110 candidate sRNAs expressed by A. baumannii during ESP growth and in the pooled growth 306 conditions (Table S3). As an example of sRNA annotation, mapped sequence reads of eight sRNAs 307 is depicted in figure 3A. The majority (74/110; 67%) of sRNAs were in intergenic regions. The 3′ 308 regions of coding genes are increasingly recognized as genomic locations that can harbor sRNA 309 genes (5, 38, 39). In A. baumannii, we identified 22 sRNAs that mapped to the 3′ regions of coding 310 genes, all of which possess their own promoters located within the upstream coding gene. We 311 anticipate that there will be additional regulatory sRNAs produced by endo-nucleolytic processing 312 of an mRNA, as observed in other bacteria (39). However, additional experiments are required to 313 annotate such sRNAs effectively (39). Only a small number (14/110; 13%) of sRNAs were located 314 antisense of coding genes. 315

We used Northern blots to validate sRNA annotations (Fig. 3). Sequence-specific 316 riboprobes were designed to hybridize to two families of homologous sRNAs and five randomly 317 selected unique sRNA candidates, including one sRNA (sRNA17) located in the 3′ region of 318 ACX60_03050, a predicted TonB-dependent receptor protein. Eighteen of the 110 sRNA 319 candidates were homologs (i.e. present as 18 copies with highly similar nucleotide sequence), 320 which we classified as “group I sRNAs” (Fig. 3B). Gene duplications are common amongst sRNA 321 species and may benefit the bacterium by allowing for rapid evolution and diversification of gene 322 regulation (34). The transcriptomic data suggested a variable length of the group I sRNAs and 323 Northern blotting with a riboprobe hybridizing to the homologous region of group I sRNAs 324 displayed multiple bands between 100-250 nt in length. The group I family of A. baumannii ATCC 325 17978 sRNAs corresponds to the “C4-similar” group in A. baumannii 5075, reported previously 326 to share regions of homology with bacteriophage P1 and P7 (37). Two of the group I A. baumannii 327 ATCC 17978 sRNAs contained a 5′ region with low sequence homology; the diversity in this 328 region may provide these particular sRNAs with additional species-specific functions (Fig. 3B). 329

We next considered orthologs of sRNA that have been characterized in model species like 330 E. coli. sRNA37 (119 nt) is predicted to be the 4.5S RNA of the signal recognition particle that is 331 involved in membrane protein targeting. sRNA84 (182 nt) is predicted to be the 6S RNA that can 332 bind and inhibit RNA polymerase in E. coli (4). 333

The RNA binding proteins Hfq, CsrA or ProQ coordinate binding of sRNAs to target 334 mRNAs in Enterobacteriaceae (40-42). Of these three sRNA-binding proteins, only Hfq has been 335 studied functionally in Acinetobacter spp. and only in A. baylyi. A. baylyi Hfq possesses an 336



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unusually long, glycine-rich C-terminus that could alter Hfq function compared to what has been 337 observed in model species (43). Thus, post-transcriptional regulation by Hfq and other RNA-338 binding proteins should be explored in Acinetobacter species. 339

340

Conservation of Acinetobacter small RNAs 341

A gene conservation analysis was carried out by comparing the nucleotide sequence identity of the 342 sRNAs identified in 17978-mff with 26 related bacterial genomes. The suite of 27 genomes 343 contained fifteen A. baumannii strains, seven non-baumannii Acinetobacter species and five more 344 distantly related members of the Pseudomonadales order (Fig. 4). The most highly conserved 345 sRNA across all species were 4.5S RNA (sRNA37), 6S RNA (sRNA84) and to a lesser extent 346 tmRNA (sRNA89). The conservation of A. baumannii sRNAs was much higher in the 347 opportunistic pathogens A. pittii and A. nosocomialis showing a mean sequence identity across all 348 110 sRNAs of ~87% for A. pittii and ~84% for A. nosocomialis compared to other Acinetobacter 349 species (>70% mean sequence identity across 110 sRNAs). Environmental, non-pathogenic 350 Acinetobacter species had lower mean sequence identity. Among the bacterial strains analyzed, 351 eleven sRNAs were highly specific to A. baumannii (with less than 70% sequence identity across 352 non-A. baumannii strains) and five sRNAs were only present in 17978 (sRNA11, sRNA23, 353 sRNA52, sRNA82 and sRNA83). None of the 110 sRNA were highly conserved (> 75% sequence 354 identity) in the more distantly related Moraxella or Pseudomonas species. We speculate that 355 sRNAs conserved in A. baumannii, but absent from other species, may have evolved for roles in 356 A. baumannii virulence or antibiotic resistance that future studies might address. The biological 357 functions of Acinetobacter sRNAs may not easily be predicted due to the low level of sequence 358 conservation between Acinetobacter sRNAs and well-characterized sRNAs from relatively 359 closely-related Pseudomonas species. 360

Functional genomics for the study of virulence and antimicrobial resistance in A. baumannii 361

A. baumannii is responsible for an increasing number of hospitalized cases of pneumonia leading 362 to death, which has fueled efforts to identify genes that are important for virulence, host 363 colonization and antimicrobial resistance. In a mouse model of A. baumannii pneumonia, genome-364 wide transposon mutagenesis followed by DNA sequencing (insertion sequencing) identified 157 365 genes associated with A. baumannii persistence in the lung confirming the importance of known 366 virulence genes and identifying several genes with no previous connection to A. baumannii 367 infection (44). Our dataset builds upon this analysis by offering detailed molecular insights into 368 the transcriptional architecture of important A. baumannii virulence factors. Here, we provide 369 examples of how AcinetoCom can be used by researchers to evaluate transcriptional features of 370 both well-characterized and novel virulence factors, and discuss the importance of in vitro growth 371 conditions in the study of virulence gene expression (Table S4). 372



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Regulation of Zinc Acquisition and the Znu ABC Transporter 373

Metal acquisition genes are a critical component in A. baumannii virulence as they are required to 374 wrest essential co-factors from host chelators (4, 45, 46). Zinc limitation has been demonstrated 375 as a central feature in controlling bacterial replication in the lung and dissemination to systemic 376 sites in the murine model of A. baumannii pneumonia (45, 47). In A. baumannii, the Zur (zinc 377 uptake regulator) transcriptional regulator is responsible for mediating the expression of several 378 zinc-associated functions, including the repression of an ABC family zinc transporter encoded by 379 znuABC. In A. baumannii 17978, the zur gene is co-transcribed as part of an operon with znuCB, 380 which encode the cognate ATPase and permease protein of the ABC transporter (45). The third 381 component, znuA, is found on the opposite DNA strand and encodes a substrate-binding protein 382 (45). During growth in zinc-replete conditions, the Zur protein binds to a 19-bp operator site 383 located 30 nucleotides upstream from the znuA translation start site and represses the expression 384 of both znuA and the zurznuCB operon (45, 47). 385

TSSpredator identified a total of 8 TSS within the chromosomal region that includes both 386 the znuA gene and zurznuCB operon (Fig. 5A). Two of the TSS were located 29 and 93 nucleotides 387 upstream of the znuA ORF. While both TSS were annotated as antisense to the zur gene, their 388 location within the intergenic region also make them prime candidates as primary or secondary 389 TSS to znuA. No nearby TSS were predicted for the zurznuCB operon; however, manual inspection 390 revealed potential primary and secondary TSS located 21 and 78 nucleotides upstream that were 391 observable but not enriched in the TEX sample relative to the RNApool sample. Based on these 392 predictions, the TSS for znuA or zurznuCB are found on either side of the Zur-binding site and 393 may represent an important regulatory feature for Zur-based transcription repression (45, 47). An 394 additional TSS was identified within the znuC ORF, located 239 nucleotides upstream of znuB. To 395 our knowledge, this TSS has not been previously identified in the well-characterized zurznuCB 396 operon and suggests the potential for transcription of znuB independently from other members 397 within the operon. Such a format of expression may be a mechanism for cells to accommodate 398 modest increases in the intracellular level of zinc while avoiding simultaneous increases in the Zur 399 protein. Further, there are no reports of a Zur box associated with the region surrounding this 400 internal TSS. A Rho-independent transcriptional terminator is also present in this region, located 401 within the znuB ORF or 113 nucleotides downstream of znuC. Several additional internal TSS 402 were also identified within the znuA gene, but did not demonstrate a clear association with 403 downstream genes or with an sRNA transcript. The remaining TSS within the region were 404 annotated as antisense TSS and contribute to the overall theme of pervasive low-level antisense 405 transcription within the A. baumannii transcriptome. 406

The Membrane Lipid Asymmetry (Mla) Transport System 407

Several of the genes responsible for A. baumannii persistence in the mouse pneumonia 408 model were identified as belonging to efflux pump systems (44). The authors highlighted the Mla 409 ATP-binding cassette (ABC) transport system because this system was annotated as conferring a 410 toluene tolerance phenotype (48), raising the question of why resistance to organic solvents may 411



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be important during infection of a mammalian host. However, recent studies have since uncovered 412 a central role for Mla-mediated transport in maintaining the integrity of the outer membrane of 413 Gram-negative bacteria (49, 50). Wang et al. reported a 15-fold decrease in in vivo persistence of 414 mutants lacking mlaA (44), which correlates with roles for the Mla transport system in iron 415 limitation and outer membrane vesicle formation as well as resistance to antibiotics such as 416 doripenem and colistin (51-53). 417

Our transcriptomic data reveal that genes encoding the Mla ABC transporter components 418 and periplasmic binding protein are co-transcribed in the mlaFEDCB operon (Fig. 5B). Similar to 419 the zurznuCB operon, TSSpredator did not call a TSS upstream of this operon, but manual 420 inspection of the TEX track revealed a single TSS located 29 nt upstream of mlaF at position 421 419,650. We noted greater transcript abundance of mlaDCB (most notably mlaCB) compared to 422 mlaFE in ESP, an expression pattern that was also observed for an LPS-deficient mutant of A. 423 baumannii strain 19606 (51). As there is no additional promoter within the operon, this suggests 424 that the mlaDCB portion of the transcript may turnover more slowly or is protected from 425 degradation resulting in elevated production of the periplasmic substrate binding domain MlaC. A 426 rho-independent terminator was identified downstream of the operon and provides a potential 3’ 427 border for the transcript; however, multiple rho-independent terminators were also identified 428 within the operon, with two terminators present early in the mlaF and mlaE ORFs. Like E. coli 429 and other Gram-negative bacteria, the A. baumannii mlaA homolog is found at a separate location 430 on the chromosome (Fig. 5C) and we identified a single TSS for mlaA, located 74 nt upstream of 431 the start codon. An additional antisense TSS was also identified, located opposite of the 3’ end of 432 the mlaA ORF. Both the mlaFEDCB and mlaA loci were expressed during ESP, suggesting that 433 this growth condition could be used for further investigation of the transport system. 434

Chloramphenicol resistance is regulated by transcription initiation 435

We discovered that A. baumannii 17978 cultured in M9 medium loses resistance to 436 chloramphenicol (Fig. 6). All A. baumannii strains contain the chromosomally-encoded efflux 437 pump CraA, for chloramphenicol resistance Acinetobacter. Efflux by CraA appears to be highly 438 specific to chloramphenicol, conferring a minimum inhibitory concentration >256 µg mL-1 439 chloramphenicol (54). Inhibition of CraA in A. baumannii strain 19606 increases sensitivity by 440 32-fold while deletion of craA increases sensitivity by 128-fold, respectively (54). Expression of 441 craA is highly consistent between strains of multi-drug resistant A. baumannii (55), but it is not 442 known if this expression is constitutive in nature (56). In A. baylyi, a point mutation 12 bp upstream 443 of the craA start codon caused increased stability of the craA mRNA resulting in higher craA 444 expression and increased resistance to chloramphenicol (57). 445

We used dRNA-seq and quantitative PCR to examine the promoter architecture and 446 regulation of craA in A. baumannii ATCC 17978, and tested whether loss of chloramphenicol 447 resistance is coupled to altered expression of craA (Fig. 6A). dRNA-seq revealed two promoters 448 at the craA gene, the gene-proximal TSS-1 promoter (position 367,680, Table S2) and the gene 449 distal TSS-2 promoter (position 367,564, Table S2). To examine how craA expression is regulated 450



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in response to growth conditions, we used qPCR to differentiate transcripts originating at TSS-1 451 from TSS-2. Because TSS-1 is contained within TSS-2 transcripts, TSS-1 transcripts were 452 calculated by subtracting TSS-2 transcripts from total craA transcripts. During steady-state rapid 453 growth (exponential phase) in nutrient-rich L-broth, 92% of craA transcription originates at TSS-454 1 (Fig. 6A). Conversely, in the nutrient-poor medium M9 plus pyruvate, TSS-1 activity was 455 undetectable and all transcription was driven by TSS-2. Next, we tested how the two promoters 456 responded to nutrient downshift. Within four hours of transferring exponentially-growing cells 457 from L-broth to M9 plus pyruvate, TSS-1 was down-regulated by 50-fold, whereas TSS-2 was 458 down-regulated only 1.4-fold (Fig. 6B). Therefore, the promoter driving TSS-1 is highly 459 responsive to nutritional quality of the growth medium. Over the following days, both promoters 460 continued to decline in activity. TSS-1 has a poor match to the RpoD –35 consensus, suggesting 461 that protein transcription factors control TSS-1 by recruiting RNAP to the promoter. The TSS-2 462 promoter contains an archetypical –35 sequence, suggesting that RNAP can bind this promoter 463 without assistance from an activator protein, which could explain the consistent expression of TSS-464 2 in L-broth and M9 medium. 465

We hypothesized that down-regulation of craA in M9 medium would result in a 466 chloramphenicol-sensitive phenotype akin to that of a craA mutant strain. Thus, we evaluated the 467 proportion of resistant and sensitive A. baumannii cells in liquid cultures. During exponential 468 growth in rich medium (L-broth), all cells were resistant to chloramphenicol (Fig. 6B). All cells 469 remained resistant at four hours after transfer to M9 medium, but 24 hours after transfer to minimal 470 media, 50% of cells were susceptible. Cell viability was high after 96 hours in M9 medium, but 471 chloramphenicol resistance had declined by more than 3,000-fold (Fig. 6B). Taken together, these 472 results suggest that antibiotic resistance is conditional, especially when cells are instantly deprived 473 of amino acids upon transfer from Lennox medium to M9 plus pyruvate medium. Although 474 chloramphenicol resistance is ubiquitous in A. baumannii isolates due to drug efflux by CraA, we 475 have discovered that craA expression —and thus chloramphenicol resistance— is conditional on 476 nutritional quality of the environment. This lack of endogenous activation or repression of drug 477 resistance presents a potential target for the effective treatment of multi-drug resistant infections 478 with an inexpensive and widely available antibiotic. 479

CONCLUSION 480

As an emerging pathogen in the “omics” age, A. baumannii pathobiology is defined largely by 481 high-throughput technologies that can guide molecular studies of pathogenicity mechanisms and 482 antimicrobial resistance. We have conducted the first dRNA-seq analysis of the model strain A. 483 baumannii ATCC 17978 to identify several thousand TSS at single nucleotide resolution, and 484 generated a comprehensive map of the transcriptome that includes 110 sRNAs. Although 3731 485 TSS is the largest and most precise dataset yet for Acinetobacter, it is important to note that 486 computational identification of TSS can overlook true sites. TSS are only called where a sharp 487 increase in read depth in the TEX-treated sample exceeds the normalized read depth in the Pool 488 sample. Overall, we identified an average of 5.1 sense strand TSS/10 kbp in A. baumannii ATCC 489



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17978, which is similar but fewer than the 7.0 sense strand TSS/10 kbp identified in S. 490 Typhimurium 4/74 (11). Because manual curation of S. Typhimurium 4/74 identified more TSS 491 per bp, we suspect the Predator program used here did not identify all TSS. Thus, scientists using 492 AcinetoCom are encouraged to examine the RNA-seq read depth at their gene(s) of interest. A 493 sharp increase in read depth in the TEX track alone can be highly suggestive of a TSS. Thus, 494 investigators are advised to scrutinize cases where there is high expression in the ESP tracks but 495 no TSS is called in the TEX track. 496

A growing body of evidence suggests a positive relationship between conditions that slow 497 bacterial growth and the expression of virulence factors. For notable pathogens such as Legionella, 498 Yersinia, and Salmonella, key virulence traits are induced during the early stationary phase in 499 laboratory culture (10, 58-61). Expression of virulence determinants in standard laboratory 500 conditions has permitted the study of gene function and characterization of the regulatory networks 501 that control virulence gene expression in other model pathogens (62). It was fortuitous to discover 502 that A. baumannii expresses key pathogenicity genes in standard laboratory conditions, including 503 the three loci identified in the mouse model of pneumonia (4, 44) highlighted in Figure 5. 504 Similarly, simple laboratory conditions revealed an environmental dimension to the control of drug 505 resistance in A. baumannii. Although diagnostic techniques that test for the presence of antibiotic 506 resistance genes can predict resistance, our finding of chloramphenicol sensitivity in a species that 507 is considered highly chloramphenicol resistant illustrates the importance of characterizing gene 508 expression and gene regulatory mechanisms. Reduced chloramphenicol efflux due to rapid down-509 regulation of craA expression presents two intriguing leads for combating this ubiquitous 510 resistance mechanism in A. baumannii. First, characterization of the regulatory mechanism and 511 identification of the environmental cues that stimulate or repress craA expression will raise the 512 potential to predict stages of infection and locations in the host that A. baumannii may be sensitive 513 to chloramphenicol. Second, elucidation of the transcription factors controlling craA may reveal 514 additional drug targets that could be targeted synergistically. This could enhance the effectiveness 515 of a widely available and inexpensive antibiotic that is being used effectively in the treatment of 516 other multi-drug resistant infections. 517

ACKNOWLEDGEMENTS 518

This work was supported by funding from Saskatchewan Health Research Foundation (grant 2867) 519 and Natural Sciences and Engineering Research Council of Canada (Discovery grant RGPIN-520 435784-2013) to ADSC. Saskatchewan Health Research Foundation (grant 3866) to KDM. 521

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693

FIGURES AND TABLES 694

Figure 1. Chromosomal location of coding sequences, small RNAs and transcriptional start 695 sites of A. baumannii ATCC 17978-mff. Coding sequences are depicted in blue and red (plus and 696 minus strand), small RNAs in light blue (plus strand) and light red (minus strand) and TSS in grey 697 (outer dark grey ring: TSS on the plus strand, inner light grey ring TSS on the minus strand). The 698 figure was created using Circa (OMGenomics, http://omgenomics.com/circa/). 699 700 Figure 2. Characterization of transcription start sites. (A) Schematic explaining TSS 701 categories. P: Primary TSS, S: Secondary TSS, I: Internal TSS, A: Antisense TSS, O: Orphan TSS. 702 (B) Pie chart showing the number of TSS per category. (C) Histogram showing the number and 703 length of 5′ UTRs of primary (red) and secondary (black) TSS. The inset illustrates the frequency 704 of occurrence of nucleotides around the transcriptional start site. (D) Meme-derived motifs 705 showing –35 and –10 region of A. baumannii 17978 promoters. 706



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707 Figure 3. sRNA in A. baumannii ATCC 17978. (A) Normalized, mapped sequence reads from 708 RNA-seq show the expression of sRNAs 17, 37, 75, 76, 77, 84, 99 and 100 (yellow arrows). 709 Curved arrows depict TSS identified in this study and lollipop structures are predicted rho-710 independent terminators. Northern blotting of selected sRNAs are shown to the right. RNA was 711 isolated from ESP and five µg of total RNA was loaded per lane. The sRNA sizes below the 712 individual blots have been predicted from (d)RNA-seq data. (B) Sequence alignment of Group I 713 and Group III sRNAs created with the Geneious Software (v. 8.1.8); coloured bases indicate 714 conservation in at least 50% of aligned sequences (A, red; C, blue; G, yellow; T, green). The 715 riboprobes used in Northern blotting are depicted as black bars atop the sRNA alignments. 716 717 Figure 4. sRNA conservation in representative members of the order Pseudomonadales. 718 Genomes of multiple Acinetobacter species, Pseudomonas species, and Moraxella catarrhalis 719 were compared using GLSEARCH. The colour scale shows the percentage sequence identity of 720 the 110 sRNAs compared to the reference sequence from A. baumannii ATCC 17978. 721 722 Figure 5. RNA-seq analysis of znu and mla virulence operon gene expression. Mapped, 723 normalized RNA-seq reads illustrate the quantitative measure of expression. Right-angle arrows 724 indicate TSS, which are colour-coded to correspond with the TSS categories described in figure 2. 725 The lollipop structures represent predicted rho-independent transcriptional terminators. 726 727 Figure 6. Elucidation of transcriptional control of chloramphenicol resistance. (A) 728 Normalized, mapped sequence reads from dRNA-seq of the RNA pool (upper panel). Right-angle 729 arrows indicate the two TSS identified in the promoter region of the chloramphenicol efflux pump 730 gene, craA. The lollipop structure represents a predicted rho-independent transcriptional 731 terminator. Transcript abundance was quantified for TSS-1 or TSS-2 and expressed relative to total 732 transcript abundance in cells growing exponentially in LB medium, with predicted –10 and –35 733 RNAP binding sites. (B) Growth experiment of A. baumannii in M9 over 96 hours. At the indicated 734 time points, A. baumannii cells were plated on LB agar (Total cells) or LB + chloramphenicol 735 (Resistant cells). Transcript abundance of craA mRNA originating from TSS-1 or TSS-1+2 736 measured by qPCR at 0, 4, 24 and 48 hours. 737 738 Table 1. Growth Conditions Used to Induce the Global A. baumannii Transcriptome 739 Table S1. Oligonucleotides Used in this Study 740 Table S2. Transcriptional Start Sites of A. baumannii str. 17978-mff 741 Table S3. sRNAs of A. baumannii str. 17978-mff 742 Table S4. Expression data 743



https://doi.org/10.1101/236794




https://doi.org/10.1101/236794




https://doi.org/10.1101/236794




https://doi.org/10.1101/236794




https://doi.org/10.1101/236794




https://doi.org/10.1101/236794




https://doi.org/10.1101/236794


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The primary transcriptome, small RNAs, and regulation of ... · 85 Cells were grown overnight in 5 mL Lennox (L-) broth (10 g L-1. tryptone, 5 g L-1. yeast 86 extract, 5 g L-1. NaCl;